Interactions in solutions and gels of stimuli-responsive polymer systems investigated by NMR spectroscopy

Transkript

1 Charles University Faculty of Science Study programme: Physical Chemistry mgr inż. Rafał Łukasz Konefał Interactions in solutions and gels of stimuli-responsive polymer systems investigated by NMR spectroscopy Ph.D. Thesis Supervisor: RNDr. Jiří Spěváček, DrSc. Institute of Macromolecular Chemistry AS CR Department of NMR Spectroscopy PRAGUE 2018

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3 Univerzita Karlova Přírodovědecká fakulta Studijní program: Fyzikální chemie mgr inż. Rafał Łukasz Konefał INTERAKCE V ROZTOCÍCH A GELECH NA PODNĚTY REAGUJÍCICH POLYMERNÍCH SYSTÉMŮ STUDOVANÝCH NMR SPEKTROSKOPIÍ Dizertační práce Školitel: RNDr. Jiří Spěváček, DrSc. Ústav makromolekulární chemie AV ČR, v.v.i. NMR Spektroskopie PRAHA 2018

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5 Declaration: I declare that I have written this thesis independently under the supervision of RNDr. Jiří Spěváček, DrSc. I did not submit this work, or a part of it, to obtain another university degree. To the best of my knowledge, I have cited all the sources I have used. Prague Signature

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7 A scientist in his laboratory is not a mere technician: he is also a child confronting natural phenomena that impress him as though they were fairy tales. Maria Skłodowska-Curie

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9 Acknowledgments First of all, I would like to thank to my supervisor RNDr. Jiří Spěváček, DrSc. for his support, patience, time and worth advices on NMR. I would like to thank to all my colleagues from the NMR Department at the Institute of Macromolecular Chemistry AS CR, v. v. i. for their help and support. This work would not exist without colleagues from Institute of Macromolecular Chemistry AS CR, v. v. i. who synthesized; Eva Čadová, Peter Černoch, Eliézer Jäger, Svetlana Petrova, Beata Strachota and analyzed by DLS and SEC (Peter Černoch, Eliézer Jäger), studied compounds used in this dissertation. Special thanks belong to my family for their faith and support not only during my Ph. D. studies.

10 Table of Contents 1 Introduction NMR Spectroscopy NMR Experiments Stimuli-responsive polymers Temperature responsive polymers Other stimuli responsive polymers 17 2 Aims of the study List of publications included in the thesis Summary of the results PEOX homopolymers and gradient copolymers (publication 1) PEOX block copolymers (publication 2) PNIPAm block copolymers (publications 3-5) PNIPAm/CLAY hydrogels (publications 6 and 7) PVME/additives (publication 8) ph and ROS responsive polymers (publications 9-11) Summary References Appendix List of other publications not included in the thesis

11 Abstract Stimuli-responsive (stimuli-sensitive, intelligent, or smart) polymers are polymer materials which, after small external stimuli, evidently change their physical or chemical properties. Smart polymers can be classified according stimuli they respond to such as: temperature changes, mechanical stress, light irradiation, ultrasonic treatment, application of external magnetic as well as electric field, changes of ph, ionic strength, addition of the chemical agents and presence of biomolecules and bioactive molecules. Stimuli-responsive synthetic polymer systems has attracted considerable attention due to wide range of applications, i.e. controlled drug delivery and release systems, diagnostics, tissue engineering and smart optical systems, as well as biosensors, microelectromechanical systems, coatings, and textiles. Among the types of stimuli for this dissertation temperature, ph and reactive oxygen species (ROS) responsive polymer systems were studied. In case of thermoresponsive polymers, when polymer chains are molecularly dissolved in a good solvent, changes (increasing or decreasing) of temperature result in insolubility (globular nanoparticles formation) of polymer chains, called temperature induced phase-separation. ph responsive polymers change properties such as: solubility, volume (gels), chain conformation as well as which bonds can cleavage upon changes in ph of environment. The ROS responsivity results in changes in solubility, hydrolysis, phase transition, and/or degradation of polymer chains. In this work 1 H NMR spectroscopy was applied for structure and temperature induced phase transition characterization (during gradual heating and/or cooling) of various thermoresponsive polymer systems based on: poly(2-ethyl-2-oxazoline), poly(nisopropylacrylamide), poly(vinyl methyl ether), as well as for study of the: interactions between the reaction mixture components, behavior of reaction mixture during the cooling processes (to freezing) and for following the course of polymerization reaction in situ in poly(n-isopropylacrylamide)/laponite XLS cryogels. Moreover 1 H (and/or 13 C) NMR were used for polymer structure and degradation products characterization of novel MPEO-PCL diblock copolymers with acid labile ketal group as a block linkage (ph responsive), pinacoltype boronic ester self-immolative and biodegradable polyoxalate prodrug based on the anticancer chemotherapeutic hormone analog diethylstilbestrol (ROS-degradable). For thermoresponsive polymers 1 H spin-spin relaxation times (temperature and time dependences) were measured to follow the changes in interactions and molecular motions of polymer, water and/or additive in solution. Additionally, in case of copolymers for characterization of conformational changes of polymer chains 2D NOESY spectra were recorded. Keywords: stimuli-responsive, phase transition, temperature, ph, ROS, copolymers, cryogel, PNIPAm, PEO, PVME, PEOx, PCL, 1 H NMR, 1 H spin-spin relaxation times, 2D NOESY 2

12 Abstrakt Na podněty reagující (na podněty citlivé nebo inteligentní) polymery jsou polymerní materiály, které na malé vnější podněty očividně mění své fyzikální nebo chemické vlastnosti. Inteligentní polymery lze klasifikovat podle podnětů, na které reagují, jako jsou změny teploty, mechanické namáhání, ozáření světlem, aplikace ultrazvuku, aplikace vnějšího magnetického či elektrického pole, změny ph, iontové síly, přidání chemických činidel a přítomnost biomolekul či bioaktivních molekul. Na podněty reagující systémy syntetických polymerů přitahují značnou pozornost díky širokému spektru aplikací, jako jsou systémy pro řízené dodávání a uvolňování léčiv, diagnostiku, tkáňové inženýrství a "inteligentní" optické systémy, stejně jako biosenzory, mikroelektromechanické systémy, nátěry a textilie. V této disertaci byly studovány polymerní systémy reagující na teplotu, ph a reaktivní kyslík (ROS). V případě termoresponsivních polymerů, kdy jsou v dobrém rozpouštědle polymerní řetězce molekulárně rozpuštěny, se změnami teploty (zvýšení nebo pokles) dochází k teplotouindukované fázové separaci a tvorbě globulárních nanočástic. Polymery reagující na ph mění své vlastnosti, jako jsou rozpustnost, objem (gely), konformace řetězce, jakož i vazby, které se mohou při změnách ph štěpit. ROS-responzivita může vést ke změnám rozpustnosti, k hydrolýze, fázovému přechodu a/nebo degradaci polymerních řetězců. V této práci byla 1 H NMR spektroskopie použita k strukturní charakterizaci u teplotou-indukovaného fázového přechodu (při postupném ohřevu a/nebo chlazení) u různých termoresponsivních polymerních systémů na bázi poly(2-etyl-2-oxazolinu), poly(nisopropylakrylamidu) a polyvinylmetyléteru, jakož i ke studiu interakcí mezi složkami reakční směsi, chování reakční směsi během ochlazování (vymražování) a ke sledování polymerizační reakce in situ u systemů poly(n-isopropylakrylamid)/laponit XLS. Kromě toho 1 H (a/nebo 13 C) NMR spektroskopie byla využita i k charakterizaci polymerních struktur a degradačních produktů nových dvoublokových kopolymerů MPEO-PCL s labilní ketalovou skupinou tvořící spojku mezi bloky (ph-citlivé), polymerů pinakolového typu boronového esteru a biodegradovatelných polyoxalátů na bázi protinádorového chemoterapeutického hormonového analogu diethylstilbestrolu (ROS-responzivní). Ke sledování změn v interakcích a molekulárních pohybech polymeru, vody a/nebo přísady byly u roztoků termoresponsivních polymerů měřeny 1 H spin-spinové relaxační doby (teplotní a časové závislosti). V případě kopolymerů byla k charakterizaci konformačních změn polymerních řetězců měřena též 2D NOESY spektra. Klíčová slova: na podněty reagující polymery, fázový přechod, teplota, ph, ROS, kopolymery, kryogel, PNIPAm, PEO, PVME, PEOx, PCL, 1 H NMR, 1 H spin-spinové relaxační doby, 2D NOESY 3

13 LIST OF ABBREVIATIONS APS D 2 O DEB DLS DMF DMSO EOx IMK LCST MEK MOx NIPAm NMR NOESY NPs PBS PCL PEO PEOx PMOx PNIPAm PVME ROS SEC T 2 TBMK TEMED ammonium persulfate deuterated water diethylstilbestrol dynamic light scattering dimethylformamide dimethyl sulfoxide 2-ethyl-2-oxazoline 3-methyl-2-butanone lower critical solution temperature methyl ethyl ketone 2-methyl-2-oxazoline N-isopropylacrylamide Nuclear Magnetic Resonance Nuclear Overhauser Effect Spectroscopy nanoparticles Phosphate-buffered saline poly(ε-caprolactone) poly(ethylene oxide) poly(2-ethyl-2-oxazoline) poly(2-methyl-2-oxazoline) poly(n-isopropylacrylamide) poly(vinyl methyl ether) reactive oxygen species size-exclusion chromatography spin-spin relaxation time t-butyl methyl ketone N,N,N,N -tetramethylethylenediamine 4

14 1 Introduction 1.1 NMR Spectroscopy From the first observation of proton magnetic resonance in water and in paraffin at 1940s 1,2 Nuclear Magnetic Resonance (NMR) spectroscopy becomes one of the most important analytical method and excellent tool not only for the chemical structure characterization, but also for investigation of molecular interactions, as well as molecular motions of plenty of studied materials, inter alia thermoresponsive polymers 3. NMR spectroscopy takes advantage of the magnetic properties of different nuclei to provide information on molecular structure. Only the nuclei which possess non-zero nuclear magnetic momentum are suitable for NMR spectroscopy. When this kind of nuclei will be placed in strong external magnetic field they align themselves relative to the field in quantized number of orientations. Each of them corresponds to the same number of energy levels involved. The transitions between energy levels can occur by applying magnetic field with correct frequency ν resulting from the resonance condition (see Fig. 1.1) 4 ; Fig. 1.1 Representation of the precession of the magnetic momentum about the axis of applied magnetic field B 0. where the term γ is the gyromagnetic ratio, which is a constant value characteristic for the particular nucleus, and B 0 is applied external magnetic field. Gyromagnetic ratio is a crucial parameter for NMR detection sensitivity, higher value of γ results in better observation. The importance of NMR spectroscopy is based on its ability to recognize a particular nucleus according its environment in the molecule. This is related to the fact that applied magnetic field B 0 induces electron cloud surrounding the nucleus, and the local field of nucleus is 5

15 different than the applied field. This effect is called magnetic shielding, and nuclei with different chemical environment are differing in the value of resonance frequency. The most popular and widely used nuclei in NMR measurements are: 1 H, 13 C, 15 N, 19 F, 29 Si and 31 P. The majority of them has spin one half, and they are frequently main components of organic or macromolecular compounds. Important factor related to effective sensitivity of NMR measurements is natural abundance of NMR visible isotope. For example, by taking into account natural abundance and molar receptivity of 1 H (99.98%, 1) and 13 C nucleus (1.07%, ); 1 H is around 5700 times more sensitive than 13 C, which leads to different times required to record good quality NMR spectra of those nuclei. The chemical shift δ is the variation of resonance frequency with shielding and can be expressed as a difference between the resonance frequency of the sample ν s and frequency of reference compound ν ref, as follows: δδ = νν ss νν rrrrrr νν rrrrrr 10 6 The chemical shift is dimensionless quantity always given in parts per million (ppm). The values of chemical shift are typical for different types of chemical groups where a lower field resonance = higher value of chemical shift. Second important information about molecular structure is obtained from indirect spin-spin coupling ( n J-coupling). This phenomenon describes the interaction between nuclei via n chemical bonds. Basically this is an influence of neighboring spins on multiplicity of peaks in the spectrum. The coupling constant J which describes the measure how powerfully nuclear spins influence each other, is a distance between the two peaks for the resonance of one nucleus split by another, and is measured in hertz (Hz). Values of J represent an interaction through bonds (2-5 covalent bonds in homonuclear proton coupling), and are independent of external magnetic field. J-coupling is a helpful parameter to gain information about bond strengths or steric arrangements. The integral intensity of the signal is the area under the signal curve. By comparison of the intensities in a spectrum, the ratios of the protons in the molecule are obtained. Signal intensities are important in structure determination, as well as to make possible quantitative analysis of the mixtures. In macromolecular chemistry integral intensities are widely used in molecular weight determination (end-group analysis), copolymer composition, and polymer chain structure or conformation characterization. The nuclear Overhauser effect (NOE) arises from direct through-space magnetic interactions between nuclear spins. The NOE is using a transfer of nuclear spin polarization 6

16 from the one Boltzman nuclear spin population distribution to the other one, by crossrelaxation. No J coupling need to be present between the nuclei. This effect has great utility in three-dimensional structure determination, and can be detected between nuclei in distance in space less than 5 Å NMR Experiments D NMR Experiments 1 H NMR The vast majority of organic compounds such as polymers contain hydrogen atoms. Observation of transitions between magnetic energy levels of the most widespread (99.98%) hydrogen isotope 1 H (spin number I = ½) is done by 1 H NMR spectroscopy. Nowadays proton NMR spectroscopy belongs to the most welcome and frequently used instrumental analytical method in structural analysis of organic compounds. The advantages of the method are: easiness of sample preparation, low concentration of the sample and short time of measurements (usually few minutes). The result of 1 H NMR experiment (single pulse sequence) is spectrum with measuring range 15 ppm (see Fig. 1.2). Fig. 1.2 Representation of proton chemical shifts 4. In general 1 H NMR spectrum can be divided in three main regions. Starting from standard tetramethylsilane (TMS) with chemical shift equal to zero, aliphatic protons signals are observed till around 2 ppm. Second region ( 2-5 ppm) is covered by signals of protons with electronegative atoms neighborhood (O, N, etc.). The low-field area of the spectrum ( 5-12 ppm) is related to signals of protons attached to unsaturated groups (C=C, C=O, aromatic). 7

17 13 C NMR The direct observation of carbon skeleton of organic compounds is possible by 13 C NMR spectroscopy measurements. In comparison to 1 H NMR, 13 C carbon isotope has low natural abundance (1.07%), and 13 C NMR experiment requires much more scans (long time of experiment). Typical 13 C NMR measurement is provided by using single pulse sequence with proton decoupling (to avoid splitting of carbon signals). Due to fact that 13 C NMR spectrum has broader range ( 220 ppm, see Fig. 1.3) than proton one, probability of peaks overlapping is smaller. Similar to proton NMR, 13 C NMR spectrum can be divided in three regions with dependency described above. Fig. 1.3 Representation of carbon chemical shifts D NMR Experiments In two-dimensional (2D) NMR both abscissa and ordinate are frequency axes. The first frequency dimension is direct measurement of frequency, and in second frequency dimension, magnetic interactions between nuclei through structural connectivity (COSY, HMBC, HSQC), spatial proximity (NOESY, ROESY), or kinetic exchange (EXSY) are shown. 2D NOESY The Nuclear Overhauser Effect Spectroscopy (NOESY) is showing correlation between protons close in space to each other by using their homonuclear NOE interactions. At NOESY spectrum diagonal signals represent 1 H NMR spectrum, and cross-peaks are related to pairs of protons with spatial proximity in limited distance 5 Å. The cross-peaks intensity decreases with a six power distance between nuclei. The NOESY spectra are extremely valuable to clarify conformational problems of macromolecules. 8

18 Relaxation Experiments In the NMR experiment radio frequency pulse is applied, and by this action thermal equilibrium of the spin system is disturbed. Following this action, the population ratios are changing, and transverse magnetic field components (M x and M y ) appears. When the perturbation stops, the system relaxes to re-establish equilibrium condition. They are two relaxation processes: first is characterized by time constant spin-lattice (longitudinal) relaxation time T 1, the relaxation in the applied field direction, and second one is characterized by time constant spin-spin (transverse) relaxation time T 2, relaxation perpendicular to the field direction. A measurement of relaxation times gives important information about mobility in studied compounds. Spin-spin Relaxation (T 2 ) The spin-spin relaxation time determines the decay of the x, y magnetization and is related to the line-width. Spin-spin relaxation time T 2 constant characterizes molecular mobility which depends on size and/or interactions between solute and solvent. Temperature as well as time measurements allow us to characterization of interactions changes caused by time or some external stimuli. The measurement of spin-spin relaxation time T 2 is carried by using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence (see Fig. 1.4 left) 5. Fig. 1.4 The diagram of Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence (left), and time domain spectrum (right) 5. The x and y are 90º x and 180º y pulses, and τ is a delay between 180º y pulse repeating n times. T 2 can be determining by performing series of experiments with increasing 2τn (i.e. 2τ, 4τ, 6τ etc.) and record data after the last even echo peak in each case. The decay in intensities of these echoes is determined only by spin-spin relaxation (see Fig. 1.4 right). In solids, viscous liquids, or large molecules in solution T 2 relaxation is fast (short T 2 values) and gives broad signals. In these cases T 2 values can be determined from the half-height linewidth Δν 1/2, by using the relation 5 ; νν1/2 = 1 ππtt 2 9

19 1.2 Stimuli-responsive polymers Polymer materials which, after small external stimuli, significantly change their physical or chemical properties, are called stimuli-responsive (stimuli-sensitive, intelligent, or smart) polymers 6 9. Stimuli-responsive polymers can be classified according to their physical forms, as well as by the types of stimuli they respond. Based on physical forms these polymers can be divided into: polymeric solutions, covalently crosslinked and physical gels, surfaces and interfaces, and also polymeric solids The types of stimuli can be systematized in three categories of responses: physical, chemical and biochemical (biological) 17. The physical stimuli is caused by external effects such as temperature changes, mechanical stress, light irradiation, ultrasonic treatment, or application of external magnetic as well as electric field Chemical stimuli includes: changes of ph, ionic strength, or addition of the chemical agents A biochemical stimulus contains presence of biomolecules and bioactive molecules such as enzymes, antigens/antibodies, proteins and glucose 26,27 (see Fig 1.5). The changes cause by the stimuli depends on its type and can be either reversible or irreversible. In addition one stimuli can result in more than one response, and also after multiple stimuli follows one or more responses 28,29. Furthermore, different responses are expected for various types of stimulus, or physical state. Examples of such responses are connected with changes in polymer-polymer, polymer-solvent interactions (ionic, hydrophobic, van der Waals and hydrogen bonding), conformational changes, micellar formation, swelling/collapsing, change in shape, simple chemical reactions (cross-linking, degradation) 14,

20 Fig. 1.5 Schematic representation of stimuli-responsive polymers classification according: physical state (left), and types of stimuli (right). Design and development of stimuli-responsive polymers has great interest due to the wide range of applications such as: control drug and gene delivery systems, bioseparation, sensors and actuators, tissue engineering, chemical valves, membranes with controlled porosity, electronic systems, diagnostics, optical systems, artificial muscles, coatings, textiles, as well as, biocatalysts and gas oil industry 7 12,17, From the aforementioned stimuli, in the following, temperature as a most frequently used stimulus will be discussed. Among others ph and reactive oxygen species (ROS) responsive polymers will be briefly reviewed Temperature responsive polymers Among the miscellaneous stimuli aforementioned, due to non-invasive application, temperature is the most extensively employed. Moreover, thermoresponsive behavior of polymer solutions, gels, and surfaces is often completely reversible It is well known that 11

21 polymers in solution are sensitive to changes in the temperature, which mostly results in coil size variations 54. Therefore, according the aforementioned definition of stimuli-responsive polymers, temperature responsive polymers are only those which considerably change their properties with small change in temperature. These changes are typically coil - globule transition in solutions, and volume transition in gels 31,55. In case of thermoresponsive polymers, when polymer chains are molecularly dissolved in a good solvent, changes (increase or decrease) of temperature result in insolubility (precipitation) of polymer. In other words, the binary polymer/solvent mixture undergoes a temperature induced phase separation from one-phasic to the bi-phasic system (two phases in equilibrium) 56,57. They are two main types of thermoresponsive polymers; the first, when phase separation occurs as a result of increasing in temperature of solution, system exhibits a lower critical solution temperature (LCST) behavior, and second is the reverse case, when phase separation is observed during decreasing temperature, which is called upper critical solution temperature (UCST) type behavior. As is shown at Figure 1.6 LCST and UCST are critical temperature points of phase separation. Fig. 1.6 Schematic illustration of phase diagrams for polymer solutions: (a) lower critical solution temperature (LCST) behavior, and (b) upper critical solution temperature (UCST) behavior. Non-ionic polymers, which exhibit LCST behavior in water, have great interest due to numerous applications. Polymers of this type are hydrophilic and very well soluble at low temperature, but heating results in hydrophobicity of polymer chains. Below the transition temperature of the solution, polymer chains are hydrated, it means that they are forming hydrogen bonds with surrounding molecules of water. Those hydrogen bonds become weaker with increasing temperature, and water molecules are releasing from the polymer structure. 12

22 The effect of these changes is dehydration and agglomeration of polymer chains called coil to globule transition of thermoresponsive polymer (Fig. 1.7). From the thermodynamic point of view, using the Gibbs equation ΔG = ΔH TΔS ( G- Gibbs free energy, H- enthalpy, S- entropy, T- temperature), below LCST hydrogen bonding between water and polymer gives favorable enthalpy contribution due to enhancement ordering of water molecules which interact with the polymer. Consequently, it contributes unfavorably to the entropy of mixing. With increasing of the temperature entropy term becomes prevalent, free energy of mixing gets positive, which is demonstrated in phase separation. In other words, entropy of the water is increasing (water is less ordered when hydrogen bonds between polymer and water are broken) and this is main driving force of the process. This phenomenon is also called hydrophobic effect 44, Fig. 1.7 Schematic representation of LCST type coil to globule phase transition, in macroscopic scale observed by turbidity of polymer solution. Many types of water soluble polymers exhibit LCST type behavior 18,61,62 (examples are shown at Fig. 1.8), according chemical composition those polymers can be divided for three main groups: polymers bearing amide groups, poly(ether)s and phosphorus polymers 60,62. The most known and widely studied thermoresponsive polymer is poly(nisopropylacrylamide) (PNIPAm), LCST behavior of PNIPAm was firstly reported in , with very sharp transition (with hysteresis during cooling process) and transition temperature around 32 C LCST of PNIPAm is close to human body temperature and can be tuned by incorporation of hydrophilic groups or monomer units, what makes this polymer and its copolymers interesting in biomedical applications 44,68. PNIPAm belongs to poly(n-substituted (meth)acrylamide)s subgroup of thermoresponsive polymers. Other homopolymers from this group with reported LCST are: poly(n-n-propylacrylamide) (PNNPAM, LCST 10 C), poly(n-cyclopropylacrylamide) (PNCPAM, LCST 53 C), 13

23 poly(n,n-diethylacrylamide) (PDEAM, LCST 33 C tacticity dependent), poly(nisopropylmethacrylamide) (PNIPMAM, LCST 43 C), and many others Second subgroup of thermoresponsive polymers bearing amide group are poly(n-vinyl amide)s. One of the polymers from this group is poly(n-vinyl caprolactam) PVCL, which contains hydrophilic heterocyclic, seven-membered lactam ring. PVCL is non-ionic biocompatible, low toxic, organic and water soluble polymer with phase transition around 31 C. These properties of PVCL, as well as high complexing ability and good film-forming make this polymer interesting for biomedical applications 18, From the remaining homopolymers which has amide side chain with the nitrogen directly bonded to the polymer backbone is worth to mention about: poly(n-vinyl isobutyramide) (PNVIBA, LCST 39 C), and poly(nvinyl pyrrolidone) (PVPy, LCST only with the presence of salt) 82,83. Another class of thermoresponsive polymers are poly(2-alkyl-2-oxazolines) (POx) with nitrogen atom in polymer backbone. Hydrophilicity of POx can be controlled by the length of the side chain of homopolymer: poly(2-methyl-2-oxazoline) (PMeOx) is hydrophilic, thus very well soluble in water, thermoresponsive LCST behavior exhibit poly(2-ethyl-2-oxazoline) (PEOx, LCST 62 C), poly(2-isopropyl-2-oxazoline) (PiPOx, LCST 36 C), poly(2-cyclopropyl-2- oxazoline) ) (PcPOx, LCST 30 C) and poly(2-n-propyl-2-oxazoline) (PnPOx, LCST 36 C) aqueous solutions (critical temperature point depends on molar mass of polymer chains), while polymers with longer side chains are no more soluble in water. Due to their biocompatibility, POx can be used in biomedical applications 38,59, Second group of LCST type thermoresponsive polymers constitute poly(ether)s. Poly(ethylene oxide) [or poly(ethylene glycol) (PEO, PEG)], and poly(propylene oxide) [or poly(propylene glycol) (PPO, PPG)] and their copolymers are widely known for their thermoresponsive behavior, especially PEO homopolymer which at high temperatures exhibits both LCST and UCST phase separation called closed loop 59. In PEO-PPO copolymers main driving force for thermoresponsive behavior is amphiphilic balance in the chain structure. Those copolymers depending on composition exhibit cloud points in range C are known as Pluronics, Tetronics, Poloxamers and are commercially available Moreover, short oligo(ethylene glycol) can be used as a side chain. Poly(oligo(ethylene glycol) mathacrylate)s (POEGMA) aqueous solutions exhibit lower values of LCST than high molecular weight linear PEO homopolymers. For example poly(2-(2-methoxyethoxy)ethyl methacrylate) (PMEO 2 MA) with two ethylene oxide units, and poly(2-[2-(2- methoxyethoxy)ethoxy] ethyl methacrylate) (PMEO 3 MA) with three ethylene oxide units shows LCST around 26 C and 52 C respectively. Polymers with longer side chains (4-9 ethylene oxide units) exhibit phase 14

24 transitions between C. Advantage of those polymers is possibility of tuning of the LCST temperature by synthesis of PMEO 2 MA-co- POEGMA copolymers (with variety of monomers ratios) Other type of polymers from this group are poly(vinylether)s. Poly(vinyl methyl ether) (PVME) is the most known and widely studied polymer from this type. PVME exhibits phase transition temperature around C, which makes this polymer interesting for biomedical applications. Other thermoresponsive polymers from this type are poly(2-methoxy-ethyl vinyl ether) (PMOVE, LCST 70 C) and poly(2-(2-ethoxy)ethoxyethyl vinyl ether) (PEOEOVE, LCST 41 C) Among the polymers bearing phosphorus in monomer repeating unit, homoplymers with reported thermoresponsive behavior can be found. The good examples are biodegradable and biocompatible poly(ethyl ethylene phosphate) (PEP, LCST 38 C) and poly(isopropyl ethylene phosphate) (PIPP, LCST 5 C), as well as poly[(alkyl ether) phosphazens] with transitions between C Fig. 1.8 Examples of temperature sensitive polymers and their LCST. The most common experimental technics for study thermoresponsive polymers are: optical and UV turbidimetry, light scattering, X-ray and neutron scattering, calorimetry, fluorescence, IR spectroscopy, Raman spectroscopy, viscometry, dielectric spectroscopy, theoretical studies, as well as NMR spectroscopy 51,52,82,89,96, Among aforementioned methods NMR spectroscopy is giving quantitative information about the LCST phase separation behavior. NMR relaxation time and diffusion experiments can show changes in molecular motions of polymer and water in solution. 2D NOESY measurements give information about conformational changes of polymer chains. To characterize coil to globule phase separation it is required to measure series of 1 H NMR spectra during gradual heating or/and cooling processes. When polymer is thermoresponsive like PNIPAm, with increasing 15

25 temperature the spectra show a visible reduction in integral intensities of all signals related to the polymer (example for PNIPAm aqueous solution (c=5% wt) is presented at Fig. 1.9 a). This result is evidently related to the fact that with increasing temperature, the mobility of the polymer chains decreases to such an extent that they escape from detection in high-resolution NMR spectra. Next for quantitative characterization of changes occurring during the heating or/and cooling processes, the values of the fraction p for units with significantly reduced mobility can be calculated by using the relation presented at Fig. 1.9c 115, where I(T) is the integrated intensity of given polymer signal in the spectrum at given temperature T and I(T 0 ) is the integrated intensity of this signal when no phase transition or other reason for the reduced mobility of polymer segments occurs. For T 0 it is necessary to choose the temperature where the integrated intensity of the given signal is the highest and therefore p(t 0 ) = 0. Additionally, in denominator of the equation one should take into account the fact that the integrated intensities should decrease with absolute temperature as 1/T. In the last step temperature dependence of p- fraction can be obtained (Fig. 1.9b), and phase separation is described by three parameters (Fig. 1.9d): extent of separation (p max ) which gives information about how many percent of polymer chains change from coil to globular state, transition width shows temperature range in which phase separation occurs, and LCST - phase transition temperature (point at temperature axis at 0.5 of p max ). 16

26 Fig H NMR characterization of phase transition: MHz 1 H NMR spectra of PNIPAm D 2 O solution (c=5 wt%) measured at 294.3, and K under the same instrumental conditions (peak assignments with structure are above top spectrum) (a), temperature dependence of p- fraction of PNIPAm aqueous solution (c=5%wt) (CH isopropyl group) (b), p- fraction equation (c), and parameters describe phase transition (d) Other stimuli responsive polymers ph responsive polymers ph responsive polymers change properties such as: solubility, volume (gels), chain conformation as well as which bonds can cleavage upon changes in ph of environment. Due to the fact that ph values vary in the human body, for example at the tissue level, cancerous 17

27 and inflamed tissues have lower ph than healthy tissues; this type of polymers has attracted considerable attention due to potential application as drug delivery agents. Generally ph responsive polymers can be divided into three groups: acidic, basic and ph neutral. Polymers having acidic or basic groups accept or release protons with changes of ph values. Depending on their pk a values, they obtain polyelectrolyte nature at acidic or basic ph. Therefore this ionic/non-ionic transition change hydrophilicity of polymer in aqueous phase and results in reversible precipitation/solubilization of polymer chains, hydrophilic/hydrophobic behavior of polymeric surfaces and swelling/deswelling in hydrogels. ph responsive acidic group constitute polymers with carboxylic, sulfonic acid, phosphoric acid, aminoacid and boronic acid groups. Group of weak polybases includes polymers with tertiary amine, morpholino, pyrrolidine, piperazine, as well as, pyridine and imidazole groups. Additionally dendrimers such as: poly(amidoamine), poly(ethylene imine) and poly(propylene imine) can be also classified as ph responsive polymers. ph responsive neutral polymers contain in main or side chain acid-labile moieties, which usually degrade under mild acidic conditions. In most cases this kind of bonds is used as a linker between polymer and drug in polymer-drug conjugates. Drug is released in target (i.e. cancerous or inflamed tissue) by cleavage of the linker. Typical ph-labile bonds used in polymer systems are, hydrazone, acetal/ketal, imine, orthoester and others 6,23,43,67, Reactive oxygen species (ROS) responsive polymers Reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (HO ), singlet oxygen ( 1 O 2 ), super oxide radicals (O 2 ) are produced by the body as a result of many physiological processes. In healthy cells ROS are produced with low concentration and have crucial role in metabolism (i.e. cell growth, migration, apoptosis). On the other hand, higher ROS production leads to oxidative stress and inflammation events (damage of cellular DNA, protein and lipid molecules) and is correlated to cancer and other diseases. This high local concentration of ROS can be used in design and production of ROS responsive delivery systems. The ROS responsivity results in changes in solubility, hydrolysis, phase transition, as well as, degradation of polymer chains 25,32,43,45, The chemical structures of some ROS responsive systems are depicted at Figure

28

29 2 Aims of the study The aim of this study is to apply various NMR spectroscopy methods for: Characterization of solution behavior of thermoresponsive poly(2-ethyl-2-oxazoline) based homopolymers and copolymers in aqueous solutions. Study of thermoresponsive behavior of nanoparticles aqueous solutions of terpolymers containing poly(ethylene oxide), poly(2-ethyl-2-oxazoline) and poly(ε-caprolactone) blocks. Characterization of thermoresponsive behavior of aqueous solutions of block copolymers of PEO and PNIPAm. Studies of interactions and polymerization kinetics of poly(nisopropylacrylamide)/clay hydrogels. Characterization of additive effects on phase transition and interactions in poly(vinyl methyl ether) solutions. The structure determination and responsivity studies of novel ph and ROS responsive polymer systems. 20

30 3 List of publications included in the thesis Publication 1 R. Konefał, J. Spěváček, P. Černoch: Thermoresponsive poly(2-oxazoline) homopolymers and copolymers in aqueous solutions studied by NMR spectroscopy and dynamic light scattering European Polymer Journal 2018, in production Publication 2 R. Konefał, J. Spěváček, E. Jäger, S. Petrova: Thermoresponsive behaviour of terpolymers containing poly(ethylene oxide), poly(2-ethyl-2-oxazoline) and poly(εcaprolactone) blocks in aqueous solutions: an NMR study. Colloid and Polymer Science, 2016, 294, Publication 3 J. Spěváček, R. Konefał, J. Dybal, E. Čadová, J. Kovářová: Thermoresponsive behavior of block copolymers of PEO and PNIPAm with different architecture in aqueous solutions: a study by NMR, FTIR, DSC and quantum-chemical calculations. European Polymer Journal, 2017, 94, Publication 4 J. Spěváček, R. Konefał, E. Čadová: NMR study of thermoresponsive block copolymer in aqueous solution. Macromolecular Chemistry and Physics, 2016, 217, Publication 5 J. Spěváček, R. Konefał, J. Dybal: Temperature-induced phase transition in aqueous solutions of poly(n-isopropylacrylamide)-based block copolymer. Macromolecular Symposia, 2016, 369, Publication 6 B. Strachota, L. Matejka, A. Zhigunov, R. Konefał, J. Spevacek, J. Dybal, R. Puffr: Poly(N-isopropylacrylamide)-clay based hydrogels controlled by the initiating conditions: evolution of structure and gel formation. Soft Matter, 2015, 11,

31 Publication 7 B. Strachota, L. Matějka, A. Sikora, J. Spěváček, R. Konefał, A. Zhigunov, M. Šlouf: Insight into the cryopolymerization to form poly(n-isopropylacrylamide)/clay macroporous gel. Structure and phase evolution. Soft Matter, 2017, 13, Publication 8 L. Starovoytova, J. Šťastná, A. Šturcová, R. Konefał, J. Dybal, N. Velychkivska, M. Radecki, L. Hanyková: Additive effects on phase transition and interactions in poly(vinyl methyl ether) solutions. Polymers, 2015, 7, Publication 9 S. Petrova, E. Jäger, R. Konefał, A. Jäger, C. de Garcia Venturini, J. Spěváček, E. Pavlova, P. Štěpánek: Novel poly(ethylene oxide monomethyl ether)-b-poly(εcaprolactone) diblock copolymers containing a ph-acid labile ketal group as a block linkage. Polymer Chemistry, 2014, 5, Publication 10 E. Jäger, A. Höcherl, O. Janoušková, A. Jäger, M. Hruby, R. Konefał, M. Netopilik, J. Panek, M. Slouf, K. Ulbrich, P. Stepanek: Fluorescent boronate-based polymer nanoparticles with reactive oxygen species (ROS)-triggered cargo release for drugdelivery applications. Nanoscale, 2016, 8, Publication 11 A. Höcherl, E. Jäger, A. Jäger, M. Hruby, R. Konefał, O. Janoušková, J. Spevacek, Y. Jiang, P. W. Schmidt, T. P. Lodge, P. Stepanek: One-pot synthesis of reactive oxygen species (ROS)-self-immolative polyoxalate prodrug nanoparticles for hormone dependent cancer therapy with minimized side effects. Polymer Chemistry, 2017, 8,

32 4 Summary of the results 4.1 PEOX homopolymers and gradient copolymers (publication 1) In this part structural changes during temperature-induced phase transition in D 2 O solutions of poly(2-ethyl-2-oxazoline) (PEOx) and P(EOx-grad-2-methyl-2-oxazoline (MOx)) copolymers were investigated by 1 H NMR spectroscopy, 1 H spin-spin relaxation times (temperature and time dependences) and 2D NOESY at various temperatures. Herein obtained results fully reported in publication 1 (see Appendix) are briefly discussed. Two PEOx homopolymers (with M n =17200 and 5900), two P(EOx/MOx) gradient copolymers with 75/25 ratio of monomer units (M n = and 6300), and two P(EOx/MOx) gradient copolymers with monomer ratio 50/50 (M n = and 7300) were prepared by Peter Černoch using cationic ring-opening polymerization of EOx and MOx in acetonitrile, initiated by methyl p-tosylate. 1 H NMR spectra and fraction p of units with significantly reduced mobility. Figures and shows high-resolution 1 H NMR spectra of a D 2 O solution (c=5 wt%) of the PEOx-H homopolymer and P(EOx/MOx)(53/47)-H gradient copolymer measured under the same instrumental conditions at three temperatures. The assignment of resonances to various proton types is shown directly in the spectra measured at 360 K and chemical structure of polymers is shown at the figures. 1 H NMR spectra presented in Figures were measured at temperatures below the LCST (down), in the middle of the transition (middle) and above the LCST (up) of studied polymers. The most significant effect observed in the spectra is a visible reduction in integral intensities of all signals related to polymers units. This effect is evidently related to the fact that with increasing temperature, the mobility of the part of polymer segments which form globular-like structures (mesoglobules) decreases to such an extent that they escape detection in high-resolution NMR spectra. Nevertheless, this effect is much weaker in copolymer solution. 23

33 Fig MHz 1 H NMR spectra of PEOx-H homopolymer in D 2 O solution (c=5 wt%) measured at 295, 338 and 360 K under the same instrumental conditions. Fig MHz 1 H NMR spectra of P(EOx/MOx) (53/47)-H copolymer in D 2 O solution (c=5 wt%) measured at 295, 337 and 360 K under the same instrumental conditions. 24

34 Quantitative characterization of changes appearing during the heating and cooling process, can be provide from integrated intensities in 1 H NMR spectra by calculation the p- fraction values of the units with significantly reduced mobility by using equation presented at Fig. 1.9c. Temperature dependences of the fraction p of D 2 O solutions (c=5 wt %) of all investigated homopolymers and copolymers are shown in Fig Additionally for comparison in the Figure is also shown temperature dependence of the p-fraction for PNIPAM (M n = 35480) in D 2 O solution (c=5 wt %) (dashed line). In comparison with PNIPAM which exhibit sharp (transition width 2 K) and complete (maximum value of the p- fraction, p max = 1) phase transition, the transition of PEOx homopolymers is broader and around 20% (p max 0.8) of PEOx units possess high mobility and do not participate in the phase transition. Moreover, much higher (than for PNIPAM) values of LCST (defined as the temperature at p max /2) of the PEOx depends on the polymer molecular weight (higher molecular weight - lower LCST). In contrast to PEOx homopolymers, for P(EOx-grad-MOx) copolymers D 2 O solutions very broad and virtually independent of copolymer composition phase transition is observed. Values of p-fraction gradually increase from 315 K without any noticeable jump (transition width at least 45 K) and reach much lower maximum values (range of p max = ). Additionally, temperature dependences of the fraction p determined from integrated intensities of various PEOx and P(EOx-grad-MOx) signals are the same for heating and cooling processes so confirming that p-fraction relates to EOx and/or MOx units as a whole. PEOx-H PEOx-L P(EOx/MOx)(53/47)-H P(EOx/MOx)(75/25)-L P(EOx/MOx)(52/48)-L P(EOx/MOx)(75/25)-H p-fraction Temperature (K) Fig Temperature dependences of the fraction p of units with significantly reduced mobility in D 2 O solutions (c = 5 wt%) of PEOx homopolymers (PEOx-H, PEOx-L) and P(EOx-grad-MOx) copolymers (P(EOx/MOx)(52/48)-L, P(EOx/MOx)(53/47)-H, P(EOx/MOx)(75/25)-L, P(EOx/MOx)(75/25)-H) during gradual heating. Dashed line shows for comparison temperature dependence of the p-fraction for D 2 O solution (c = 5 wt%) of PNIPAM. 25

35 The reversibility of the phase transition was characterized by measurements of the sample during gradual cooling directly after heating process. Figure shows temperature dependences of the fraction p in D 2 O solutions (c=5 wt%) of the homopolymer PEOx-H (a)(similar behavior was obtained for PEOx-L homopolymer) and copolymer P(EOx/MOx)(53/47)-H (b) (similar behavior for other copolymers) during gradual heating and subsequent gradual cooling. For the PEOx homopolymer (Fig a) values of p-fraction remain almost unchanged in the whole range of temperatures of measurements during gradual cooling which means that on the molecular level the phase transition of PEOx homopolymer is irreversible. This is in contrast with the fact that the sample of PEOx-H in D 2 O was transparent after pulling out from the magnet at 295 K and results obtained by macroscopic cloud point measurements on 0.5 wt% solutions reported in literature. In case of copolymer samples it is observed reversible phase separation with some hysteresis (Fig b). Fig Temperature dependences of the fraction p of units with significantly reduced mobility in D 2 O solutions (c = 5 wt%) of PEOx-H homopolymer (a) and P(EOx/MOx)(53/47)-H copolymer (b) during gradual heating and subsequent gradual cooling. In next step of the investigation, effect of polymer concentration on transition temperatures was studied. For this purpose solutions of the PEOx-H homopolymer and P(EOx/MOx)(53/47)-H copolymer were prepared with three different polymer concentrations c = 0.5, 5 and 20 wt%. In Fig are shown the p-fraction temperature dependences in the PEOx-H (a) and P(EOx/MOx)(53/47)-H (b) solutions recorded for these concentrations during gradual heating. Similarly to 5 wt% concentration samples, all polymer proton types show the same p-fraction temperature behavior. It is clearly visible from the Fig a that for the PEOx-H homopolymer the way of transition for c = 0.5 wt% and c = 5 wt% is very similar. Completely different character of the transition shows solution with c = 20 wt% concentration. In this case higher starting value of the p-fraction at 295 K is gradually 26

36 increasing from 310 K to 335 K; next it drops to reach the minimum at 345 K and then again rise. Decreasing p-fraction values (in K) is a result of increasing mobility of polymer segments at these temperatures. PEOx-H solution (c = 20 wt%) is very viscous (jelly-like) at room temperature. We assume that at temperatures the respective physical network structure is breaking and reorganizing, and this process was confirmed by DSC measurements. Similar behavior of 20 wt% solution was also obtained for second homopolymer PEOx-L. Figure 4.1.5b shows results obtained for P(EOx/Mox)(53/47)-H copolymer in D 2 O solutions. In contrast to homopolymes these dependences are very similar; in all cases the established LCST values somewhat decrease with increasing concentration. Fig Temperature dependences of the fraction p of PEOx-H homopolymer (a) and P(EOx/MOx)(53/47)-H copolymer (b) in D 2 O solutions with three polymer concentrations (c = 0.5; 5; 20 wt%) during gradual heating. Spin-Spin Relaxation Times T 2 of Water (HDO) Molecules Information on behavior of water molecules and polymer-solvent interactions (hydration) during phase-transition in water solutions was obtained by spin-spin relaxation times T 2 measurements. Experiments were measured at temperatures based on the temperature dependence of the p-fraction. Results obtained for PEOx-L homopolymer D 2 O solution (c = 5 wt%) are shown in Fig : temperature dependence (Fig a) and time dependence at 360 K (Fig b). Results presented in Fig a show that while at temperatures below 335 K the T 2 relaxation curves were monoexponential, at T 335 K, i.e., in the transition region and above the transition, the relaxation curves were non-exponential and two T 2 components were necessary to fit experimental relaxation curves. These data show the existence of two types of water at temperatures above 335 K. First type is free water with longer relaxation times which are similar as T 2 values at lower temperatures (T 2 = 3-4 s) which corresponds to HDO molecules in solution. Second type is bound water with T 2 27

37 values which are 2 orders of magnitude shorter (T 2 = 42 ms at 335 K, T 2 = 21 ms at 360 K) which corresponds to HDO molecules bound (confined, entrapped) inside the collapsed globular structures. These values are virtually constant for both types of water molecules showing that arrangement with free and bound water is stable at least for 12 hrs (Fig b). Fig Temperature dependence (a) and time dependence at 360 K (b) of 1 H spin-spin relaxation times T 2 of HDO in D 2 O solution (c = 5 wt%) of the PEOx-L homopolymer. 2D 1 H- 1 H NOESY NMR spectra To obtain information on spatial proximity between proton groups of EOx and MOx units, on D 2 O solutions (c = 5 wt%) of P(EOx/MOx)(75/25)-L and P(EOx/MOx)(53/47)-H copolymers we measured 2D 1 H- 1 H NOESY NMR spectra at three temperatures: at 295 K (below the transition), 340 K (in the middle of the transition) and 360 K (above the transition) (Fig ). These copolymers have relatively low values of the p-fraction (p max = 0.32 and 0.44, respectively) at 360 K and therefore a major part of these copolymers is directly detected in high-resolution NMR spectra at this temperature. NOESY spectra recorded for the P(EOx/MOx)(75/25)-L sample are shown in Fig In spectrum measured at 295 K (Fig a), cross-peaks between various proton groups of EOx or MOx units were detected, as well as weaker cross-peaks between side chain CH 3 or CH 2 protons of EOx units (signals at 1.05 ppm and 2.35 ppm, respectively) and CH 3 protons of MOx units (signal at 2.1 ppm). EOx and MOx units which are in close proximity (<0.5 nm) can be both from the same chain of the copolymer, assuming random-coil conformation of copolymer chains at room temperature, and from different copolymer chains. At 340 K the cross-peaks between EOx and MOx protons disappeared and cross-peaks between various proton groups of EOx or MOx units are weaker (Fig b). In the NOESY spectrum measured at 360 K (Fig c) 28